Beneficial associations between plants and microbes are widespread in nature and have been studied extensively in the microbial-dominant environment of the rhizosphere. Such associations are highly advantageous for the organisms involved, benefiting soil microbes by providing them access to plant metabolites, while plant growth and development are enhanced through the promotion of nutrient uptake and/or protection against (a)biotic stresses. While the establishment and maintenance of mutualistic associations have been shown to require genetic and epigenetic reprogramming, as well as an exchange of effector molecules between microbes and plants, whether short RNAs are able to effect such changes is currently unknown. Here, we established an interaction between the model grass species Brachypodium distachyon (Bd, Pooideae) and the beneficial fungal root endophyte Serendipita indica (Si, syn. Piriformospora indica, Sebacinales) to elucidate RNA interference-based regulatory changes in gene expression and small (s)RNA profiles that occurred during establishment of a Sebacinalean symbiosis. Colonization of Bd roots with Si resulted in higher grain yield, confirming the mutualistic character of this interaction. Resequencing of the Si genome using the Oxford Nanopore technique, followed by de novo assembly yielded in 57 contigs and 9441 predicted genes, including putative members of several families involved in sRNA production. Transcriptome analysis at an early stage of the mutualistic interaction identified 2963 differentially expressed genes (DEG) in Si and 317 in Bd line 21-3. The fungal DEGs were largely associated with carbohydrate metabolism, cell wall degradation, and nutrient uptake, while plant DEGs indicated modulation of (a)biotic stress responses and defense pathways. Additionally, 10% of the upregulated fungal DEGs encode candidate protein effectors, including six DELD proteins typical for Sebacinales. Analysis of the global changes in the sRNA profiles of both associated organisms revealed several putative endogenous plant sRNAs expressed during colonization belonging to known micro (mi)RNA families involved in growth and developmental regulation. Among Bd- and Si-generated sRNAs with putative functions in the interacting organism, we identified transcripts for proteins involved in circadian clock and flowering regulation as well as immunity as potential targets of fungal sRNAs, reflecting the beneficial activity of Si. We detected beneficial effects of Si colonization on Bd growth and development, and established a novel plant-mutualist interaction model between these organisms. Together, the changes in gene expression and identification of interaction-induced sRNAs in both organisms support sRNA-based regulation of defense responses and plant development in Bd, as well as nutrient acquisition and cell growth in Si. Our data suggests that a Sebacinalean symbiosis involves reciprocal sRNA targeting of genes during the interaction.

中文翻译：

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一种新的植物-真菌关联揭示了共生开始时基本的 sRNA 和基因表达重编程

植物和微生物之间的有益关联在自然界中广泛存在，并且已在根际微生物占主导地位的环境中进行了广泛的研究。这种关联对所涉及的生物体非常有利，通过使土壤微生物获得植物代谢物而使它们受益，同时通过促进养分吸收和/或保护免受（a）生物胁迫而增强植物生长和发育。虽然已证明建立和维持共生关联需要遗传和表观遗传重编程，以及微生物和植物之间的效应分子交换，但目前尚不清楚短 RNA 是否能够影响这种变化。在这里，我们建立了模型草种 Brachypodium distachyon (Bd, Pooideae) 和有益的真菌根内生菌 Serendipita indica (Si, syn. Piriformospora indica, Sebacinales) 以阐明在 Sebacinalean 共生建立过程中发生的基因表达和小 (s) RNA 谱中基于 RNA 干扰的调节变化。Bd 根与 Si 的定殖导致更高的谷物产量，证实了这种相互作用的共生特征。使用 Oxford Nanopore 技术对 Si 基因组进行重测序，然后从头组装产生 57 个重叠群和 9441 个预测基因，包括参与 sRNA 生产的几个家族的推定成员。在互惠相互作用的早期阶段的转录组分析确定了 Si 中的 2963 个差异表达基因 (DEG) 和 Bd 线 21-3 中的 317 个。真菌 DEGs 在很大程度上与碳水化合物代谢有关，细胞壁降解和养分吸收，而植物 DEGs 表明（a）生物应激反应和防御途径的调节。此外，10% 的上调真菌 DEG 编码候选蛋白质效应物，包括六种典型的 Sebacinales 的 DELD 蛋白质。对这两种相关生物的 sRNA 谱的全局变化的分析揭示了几种假定的内源性植物 sRNA，它们在属于已知的参与生长和发育调节的微 (mi) RNA 家族的定殖过程中表达。在 Bd 和 Si 产生的在相互作用的生物体中具有推定功能的 sRNA 中，我们确定了参与生物钟和开花调节以及免疫的蛋白质的转录物作为真菌 sRNA 的潜在靶标，反映了 Si 的有益活性。我们检测了 Si 定殖对 Bd 生长和发育的有益影响，并在这些生物之间建立了一种新的植物互惠相互作用模型。总之，两种生物体中基因表达的变化和相互作用诱导的 sRNA 的鉴定支持基于 sRNA 的 Bd 中防御反应和植物发育的调节，以及 Si 中的养分获取和细胞生长。我们的数据表明，Sebacinalean 共生涉及相互作用期间基因的相互 sRNA 靶向。以及 Si 中的养分获取和细胞生长。我们的数据表明，Sebacinalean 共生涉及相互作用期间基因的相互 sRNA 靶向。以及 Si 中的养分获取和细胞生长。我们的数据表明，Sebacinalean 共生涉及相互作用期间基因的相互 sRNA 靶向。